Treatment of lung cancer

ABSTRACT

Disclosed are methods of treating lung cancer by administering to a human in need thereof effective amounts of FTS, or various analogs thereof, or a pharmaceutically acceptable salt thereof, optionally, in combination with a chemotherapeutic agent. Chemotherapeutic agents, and combinations thereof, for use with FTS, its analogs, or its salts are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 12/519,578, filed on Jun. 17, 2009, which application is a nationalphase entry under 35 U.S.C. §371 of International Application No.PCT/IL2007/001556, filed Dec. 17, 2007, which claims priority from U.S.Provisional Patent Application No. 60/875,915, filed Dec. 19, 2006, allof which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Lung cancer is the leading cause of cancer-related deaths in the world[Greenlee et al., CA Cancer J Clin 51:15-36 (2001)]. Only one in tenpatients diagnosed with this disease will survive the next five years.Although lung cancer was previously an illness that affectedpredominately men, the lung cancer rate for women has been increasing inthe last few decades, which has been attributed to the rising ratio offemale to male smokers. More women die of lung cancer than any othercancer, including breast cancer, ovarian cancer and uterine cancerscombined. [American Cancer Society. Cancer Facts and FIGS. 2006.Atlanta: American Cancer Society (2006)]. Despite advances in surgery,chemotherapy, and radiation therapy, survival rates have barely changedin the last decade, and long-term survival remains dramatically poor.

Lung cancers can arise in any part of the lung. Ninety to 95% of cancersof the lung are thought to arise from the epithelial, or lining cells ofthe larger and smaller airways (bronchi and bronchioles); for thisreason lung cancers are sometimes called bronchogenic carcinomas.Cancers can also arise from the pleura (the thin layer of tissue thatsurrounds the lungs), called mesotheliomas, or rarely from supportingtissues within the lungs, for example, blood vessels.

It has been established that lung cancer arises as a consequence of theaccumulation of multiple genetic changes involving critical genescontrolling cell motility, proliferation, differentiation, andapoptosis. [Sekido et al., Biochimica et Biophysica Acta 1378:F21-F59(1998)].

According to the American Cancer Society, there are two major types oflung cancer: small cell lung cancer (SCLC) and non-small cell lungcancer (NSCLC). SCLC comprises about 15% of all cancers. NSCLC, however,comprises about 85% of all lung cancers and is divided into threedistinct sub-types: squamous cell carcinoma (about 25-30% of the cases),large cell carcinomas (about 10-15%), and adenocarcinomas (about 40%).The cells in these sub-types differ in size, shape, and chemicalmake-up. These lung cancers are inclusive of bronchogenic carcinoma,bronchial carcinoids, chondromatous hamartoma, solitary pulmonarynodules, pulmonary sarcomas, undifferentiated small cell carcinoma,undifferentiated large cell carcinoma, and bronchioloalveolarcarcinomas.

Current research indicates that the factor with the greatest impact onrisk of lung cancer is long-term exposure to inhaled carcinogens. Themost common means of such exposure is tobacco smoke.

Treatment and prognosis depend upon the histological type of cancer andthe stage (degree of spread). Possible treatment modalities includesurgery, chemotherapy, and/or radiotherapy.

SUMMARY OF THE INVENTION

A first aspect of the present invention is directed to a method oftreating lung cancer. The method comprises administering to a human inneed thereof an effective amount of S-farnesylthiosalicylic acid (FTS)or an analog thereof, or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention is directed to a method oftreating lung cancer. The method comprises administering to a human inneed thereof effective amounts of S-farnesylthiosalicylic acid (FTS) oran analog thereof, or a pharmaceutically acceptable salt thereof, and achemotherapeutic agent.

The results of a first set of experiments described herein showed thatin five human cell lines commonly used in the study of lung cancer[non-small cell lung carcinoma cell lines (NSCLC), a human lung squamouscell carcinoma cell line, and a lung epidermoid carcinoma cell line)],FTS inhibited cancer cell growth.

The results of a further set of experiments described herein showed thatin a human lung carcinoma A549 cell line, FTS reversed the transformedmorphology of the cells, altered the cytoskeletal organization of thecells, and inhibited the anchorage-independent growth of cancer cellcolonies.

The results of another set of experiments described herein showed thatthe combined treatment of FTS with a chemotherapeutic agent in vitrocaused greater cell death with both drugs than treatment with eitherdrug alone in a human lung epithelial carcinoma A549 cell line.

The results of an additional set of experiments described herein showedthat administering FTS i.p. to a lung cancer cell nude mouse modelinhibited A549 and HTB-58 (SK-MES-1) tumor cell growth.

Yet another set of experiments described herein showed that thecombination of FTS with a chemotherapeutic agent in vivo caused greatercell death with the combined treatment than with either drug alone in anude mouse model.

The results of another set of experiments described herein showed thatin four human lung cancer (NSCLC) cell lines (H1734, H2030, H1975, andH3255) FTS sensitized the cells resulting in cell death.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph illustrating the inhibition of BrdU into the DNAof A549 cells (NSCLC) after incubation of the FTS-treated cells (75 μM)for 48 h, expressed as a percentage of control.

FIG. 2 are photomicrograph images of vehicle-treated (left) andFTS-treated (right) A549 cells (NSCLC) and further depicts the reductionin number of FTS-treated cells.

FIG. 3 is a bar graph illustrating the dose dependent inhibition of A549cell (NSCLC) growth at increasing concentrations of FTS (μM), expressedas a percentage of control.

FIG. 4 illustrates the results of a FACS analysis describing FTS inducedcell-cycle arrest in A549 cells (NSCLC).

FIG. 5 is a bar graph illustrating the dose dependent inhibition ofH-1299 cells (NSCLC) at increasing concentrations of FTS (μM) asdetermined by direct cell counting.

FIG. 6 is a bar graph illustrating the dose dependent inhibition of lungsquamous cell carcinoma cell line HTB-58 (SK-MES-1) cells at increasingconcentrations of FTS (μM) as determined by direct cell counting.

FIG. 7 is a bar graph illustrating the dose dependent inhibition of H23cells (NSCLC) at increasing concentrations of FTS (μM) as determined bydirect cell counting.

FIG. 8 is a bar graph illustrating the dose dependent inhibition ofHTB54 lung epidermoid carcinoma cells at increasing concentrations ofFTS (μM) as determined by direct cell counting.

FIG. 9 is a table summarizing the half maximal inhibitory concentration(IC₅₀) of FTS (μM) in each of the human lung cancer cell lines [A549,H23, HTB54, H-1299, HTB-58 (SK-MES-1)].

FIG. 10 is a series of six fluorescent microscopic images illustratingFTS-induced alterations in stress fiber (F-Actin) and focal adhesion(α-Vinculin) formation on the cytoskeleton of A549 cells (NSCLC).

FIGS. 11A-11C are typical immunoblots and quantitative analyses of theresults (means±SD of four experiments), as determined by densitometryand normalized to the level of expression of each protein. (A)illustrates the reduction in levels of K-Ras-GTP (upper panels) and ofphospho-ERK and phospho-Akt (lower panels) by FTS. (B) illustrates theunaffected levels of Rac1-GTP by FTS. (C) illustrates the inducedincrease in RhoA-GTP by FTS (*P<0.05 compared to vehicle-treatedcontrol).

FIGS. 12A-12B illustrates the inhibition of the anchorage-independentgrowth or transformation of A549 cells (NSCLC) in soft agar by FTS.Photomicrograph images (A) illustrate the DMSO-treated (control) cellsand colony formation before and after treatment with FTS (50 μM and 100μM). The bar graph (B) illustrates the inhibition of A549 cell colonyformation at increasing concentrations of FTS (0 μM, 50 μM, and 100 μM).

FIGS. 13A-13D are bar graphs illustrating (A) the effects of thecombination of FTS (40 μM) and gemcitabine (0, 100, and 200 nM) on A549cell (NSCLC) death; (B) the effects of the combination of FTS (40 μM)and doxorubicine (0, 50, and 100 nM) on A549 cell (NSCLC) death; (C) theeffects of the combination of FTS (40 μM) and cisplatin (0, 5.0, and10.0 nM) on A549 cell (NSCLC) death; (D) the effects of the combinationof FTS (40 μM) and paclitaxel (0, 2.5, and 5.0) on A549 cell (NSCLC)death.

FIGS. 14A-14D are bar graphs illustrating (A) the effects of i.p.administration of FTS alone (10 mg/kg) in A549-cell-implanted nude mousemodels; (B) the effects of i.p. administration of FTS alone (10 mg/kg)in HTB58-cell-implanted nude mouse models; (C) the effects of oraladministration of FTS alone (50 mg/kg) in A549-cell-implanted nude mousemodels; and (D) the effects of oral administration of FTS alone (60mg/kg), the effects of oral administration of gemcitabine alone, and thecombined effects of oral administration of FTS and gemcitabine inA549-cell-implanted nude mouse models.

FIG. 15 is a graph illustrating the effects of increasing concentrationsof FTS on human NSCLC cell lines H1734 and H2030 (KRAS mutations) andH1975 and H3255 (EGFR mutations).

DETAILED DESCRIPTION

Ras proteins act as on-off switches that regulate signal-transductionpathways controlling cell growth, differentiation, and survival.[Reuther, G. W., Der, C. J., Curr Opin Cell Biol 12:157-65 (2000)]. Theyare anchored to the inner leaflet of the plasma membrane, whereactivation of cell-surface receptors, such as receptor tyrosine kinase,induces the exchange of guanosine diphosphate (GDP) for guanosinetriphosphate (GTP) on Ras and the conversion of inactive Ras-GDP toactive Ras-GTP. [Scheffzek, K., Ahmadian, M. R., Kabsch, W., et al.Science 277:333-7 (1997)]. The active Ras protein promotes oncogenesisthrough activation of multiple Ras effectors that contribute toderegulated cell growth, differentiation, and increased survival,migration and invasion. [See, e.g., Downward, J., Nat. Rev. Cancer3:11-22 (2003); Shields, J. M., et al., Trends Cell Biol 10:147-541(2000); and Mitin, N., et al., Curr Biol 15:R563-74 (2005)].

FTS is a potent Ras inhibitor that acts in a rather specific manner onthe active, GTP-bound forms of H-, N-, and K-Ras proteins. [Weisz, B.,Giehl, K., Gana-Weisz, M., Egozi, Y., Ben-Baruch, G., Marciano, D.,Gierschik, P., Kloog, Y., Oncogene 18:2579-2588 (1999); Gana-Weisz, M.,Halaschek-Wiener, J., Jansen, B., Elad, G., Haklai, R., Kloog, Y., Clin.Cancer Res. 8:555-65 (2002)]. FTS competes with Ras-GTP for binding tospecific saturable binding sites in the plasma membrane, resulting inmislocalization of active Ras and facilitating Ras degradation. [Haklai,et al., Biochemistry 37(5):1306-14 (1998)]. This competitive inhibitionprevents active Ras from interacting with its prominent downstreameffectors and results in reversal of the transformed phenotype intransformed cells that harbor activated Ras. As a consequence,Ras-dependent cell growth and transforming activities, both in vitro andin vivo, are strongly inhibited by FTS. [Weisz, B., et al., supra.;Gana-Weisz, M., et al., supra.].

FTS and its analogs useful in the present invention are represented byformula I:

wherein

-   -   R¹ represents farnesyl, geranyl or geranyl-geranyl;

R² is COOR⁷, or CONR⁷R⁸, wherein R⁷ and R⁸ are each independentlyhydrogen, alkyl or alkenyl;

-   -   R³, R⁴, R⁵ and R⁶ are each independently hydrogen, alkyl,        alkenyl, alkoxy, halo, trifluoromethyl, trifluoromethoxy, or        alkylmercapto; and    -   X represents S.

The structure of FTS is as follows:

FTS analogs embraced by formula I, and which may be suitable for use inthe present invention, include 5-fluoro-FTS, 5-chloro-FTS, 4-chloro-FTS,S-farnesyl-thiosalicylic acid methyl ester (FTSME), andS-geranyl,geranyl-thiosalicylic acid (GGTS). Structures of thesecompounds are set forth below.

In some embodiments, GGTS is administered in an amount effective totreat a patient diagnosed with lung cancer.

Methods for preparing the compounds of formula I are disclosed in U.S.Pat. No. 5,705,528 (RE39,682) and U.S. Pat. No. 6,462,086. See also,Marom, M., Haklai, R., Ben-Baruch, G., Marciano, D., Egozi, Y., Kloog,Y., J Biol Chem 270:22263-70 (1995).

Pharmaceutically acceptable salts of the Ras antagonists of formula Imay be useful. These salts include, for example, sodium and potassiumsalts. Other pharmaceutically acceptable salts may be selected inaccordance with standard techniques as described in Berge, S. M.,Bighley, L. D., and Monkhouse, D. C., J. of Pharm. Sci. 66(1):1-19(1977). In preferred embodiments, however, FTS and its analogs are notadministered in the form of a salt (i.e., they are administered innon-salified form).

In some embodiments, treatment also includes administering ananti-cancer therapy which includes, for example, chemotherapy, radiationtherapy, immunotherapy or gene therapy, and combinations thereof.

In some embodiments, treatment includes administering a chemotherapeuticagent to a patient diagnosed with lung cancer. Chemotherapeutic agentsare those medications that are used to treat various forms of cancerand, particularly, lung cancer and its various forms and associatedmanifestations. Generally, these medications are given in a particularregimen over a period of weeks. In some cases, combination chemotherapymay be recommended. Methods of preparing and using chemotherapeuticagents are well-known in the art. See, e.g., Remington: The Science andPractice of Pharmacy (21st Edition), Lippincott, Williams & Wilkins,(2005).

Chemotherapeutic agents may be administered as the first line oftreatment or it may be started after a tumor is surgically resected, forexample. The agents may be administered by various methods including,oral (by mouth), injection (intramuscular or subcutaneous), intravenous(IV), intra-arterial (into the arteries, intralesional (directly intothe tumor), intraperitoneal (into the peritoneal cavity), intrathecal(into the spinal fluid), and topical (applied to the skin). A variety offactors, including the overall health, size and weight of the patient,the patient's tolerance to the treatment, and the type and stage of thecancer, will determine the type of chemotherapy used and the mode andduration of administration. Optimally, dosages for each of thechemotherapeutic agents are prescribed in accordance with currentlabeling instructions. Dosages, however, may be adjusted to satisfy apatient's needs.

Examples of chemotherapeutic agents include, but are not limited to,paclitaxel (Taxol®), docetaxel (Taxotere®), cisplatin, carboplatin(Paraplatin®), gemcitabine hydrochloride (Gemzar®), doxorubicinhydrochloride, etoposide (Etopophos®, Vepesid®), pemetrexed (Alimta®),topotecan (Hycamtin®), vinblastine (Velbe®), Vindesine (Eldisine®),vinorelbine (Navelbine®), ifosfamide (Mitoxana®), and Mitomycin. Thosemost commonly used agents to treat lung cancer include: gemcitabine,cisplatin, carboplatin, vinorelbine, paclitaxel, docetaxel, anddoxorubicin. These agents may be given in combination, for example,vinorelbine and cisplatin or carboplatin; gemcitabine with cisplatin orcarboplatin or paclitaxel; MIC (mitomycin, ifosfamide and cisplatin);MVP (mitomycin, vinblastine and cisplatin); and EC (etoposide andcarboplatin).

In some embodiments, the chemotherapeutic agent is paclitaxel (Taxol®)[5,20-Epoxy-1,2,4,7,10,13-hexahydroxytax-11-en-9-one 4,10-diacetate2-benzoate 13-ester with (2R,3S)—N-benzoyl-3-phenylisoserine], ananti-neoplastic agent isolated from the bark of the Pacific yew tree,Taxus brevifolia. Paclitaxel is an antimicrotubule antineoplastic agent.Paclitaxel promotes microtubule assembly by enhancing the polymerisationof tubulin, the protein subunit of spindle microtubules, even in theabsence of the mediators normally required for microtubule assembly(e.g., guanosine triphosphate (GTP)), thereby inducing the formation ofstable, nonfunctional microtubules. It is a colorless to slightly yellowviscous solution.

In one example, combination chemotherapy using Taxol® and cisplatin isindicated. The recommended regimen, given every 3 weeks, is Taxol®administered intravenously over 24 hours at a dose of 135 mg/m² followedby cisplatin at 75 mg/m².

In some embodiments, the chemotherapeutic agent is docetaxel (Taxotere®)[(2R,3S)—N-carboxy-3-phenylisoserine,N-tert-butyl ester, 13-ester with5β-20-epoxy-1,2α,4,7β,10β,13α-hexahydroxytax-11-en-9-one 4-acetate2-benzoate, trihydrate], an antineoplastic agent belonging to the taxoidfamily. It is prepared by semisynthesis beginning with a precursorextracted from the renewable needle biomass of yew plants. Docetaxeldiffers from paclitaxel at two positions in its chemical structure. Ithas a hydroxyl functional group on carbon 10, whereas paclitaxel has anacetate ester and a tert-butyl substitution exists on thephenylpropionate side chain. The carbon 10 functional group changecauses docetaxel to be more lipid soluble than paclitaxel. [Clarke, S.J., Rivory, L. P., Clin Pharmacokinet 36(2):99-114 (1999)]. The mainmode of therapeutic action of docetaxel is the suppression ofmicrotubule dynamic assembly and disassembly. [Lyseng-Williamson, K. A.,Fenton, C., Drugs 65(17):2513-31 (2005); Yvon, A. C., Wadsworth, P.,Jordan, M. A., The American Society for Cell Biology 10:947-959 (1999)].The docetaxel injection concentrate is a clear yellow to brownish-yellowviscous solution.

When used as a single agent therapy, a recommended dose regimen ofdocetaxel for patients is 75 mg/m² administered intravenously over 1hour every 3 weeks.

In some embodiments, the chemotherapeutic agent is a platinum-baseddrug. The platinum-based drugs useful in the practice of the presentinvention include cisplatin [cis-diamminedichloroplatinum(II)] and itsanalogs, e.g., carboplatin[diammine(1,1-cyclobutanedicarboxylato)-platinum(II)]. These drugs areknown to inflict damage on cellular nucleic acids, including DNA.Cisplatin acts by cross-linking DNA in various different ways, making itimpossible for rapidly dividing cells to duplicate their DNA formitosis. The damaged DNA sets off DNA repair mechanisms, which activateapoptosis when repair proves impossible. Methods of preparing and usingcisplatin as an anti-cancer agent are described in, for example, U.S.Pat. No. 5,562,925 and Inorg Synth 7:239 (1963).

Carboplatin differs from cisplatin in that it has a closed cyclobutanedicarboxylate moiety on its leaving group in contrast to the readilyleaving chloro groups. This results in very different DNA bindingkinetics. Methods of preparing and using carboplatin as an anti-canceragent are described in, for example, U.S. Pat. No. 4,657,927 and InorgChem Acta 46:L15 (1980). Both cisplatin and carboplatin are indicatedfor combination chemotherapy.

A recommended dosage of cisplatin for adults and children when used assingle agent therapy is 50-100 mg/m² as a single IV infusion every 3-4weeks, or 15-20 mg/m² as a daily IV infusion for 5 days every 3-4 weeks.

A recommended dosage of carboplatin in previously untreated adultpatients with normal kidney function is 400 mg/m² as a single IV doseadministered by short-term (15 to 60 minutes) infusion. Therapy shouldnot be repeated until four weeks after the previous carboplatin course,and/or until the neutrophil count is at least 2000 cells/mm³ and theplatelet count is at least 100,000 cells/mm³.

In some embodiments, the chemotherapeutic agent is gemcitabinehydrochloride (Gemzar®) [2′-deoxy-2′,2′-difluorocytidinemonohydrochloride]. The cytotoxic effect of gemcitabine is attributed toa combination of two actions of the diphosphate and the triphosphatenucleosides, which leads to inhibition of DNA synthesis. It is a whitepowder, which forms a clear solution. Gemcitabine, alone or incombination with cisplatin, is indicated for the first line treatment ofpatients with locally advanced or metastatic non-small cell lung cancer.[See, e.g., FDA REVISED LABEL—VERSION 082598; 010603; 051904; 042005;042605 for Gemzar®]. Combination chemotherapy for treatment of lungcancer (NSCLC) with gemcitabine also includes carboplatin [See, e.g.,Tassarini, D., et al., Tumori 90:54-59 (2004)] and paclitaxel [See,e.g., Kosmidis, P., J Clin Oncol. 20(17):3578-85 (2002)].

A recommended adult dose of gemcitabine (Gemzar®) as a single agent forlung cancer (NSCLC) is 1000 mg/m², given by 30-minute intravenousinfusion. This should be repeated once weekly for three weeks, followedby a one-week rest period. This four-week cycle is then repeated. Dosagereduction with each cycle or within a cycle may be applied based uponthe amount of toxicity experienced by the patient.

A recommended adult dose of gemcitabine for combination therapy usingcisplatin, for example, has been investigated using two dosing regimens.One regimen used a three-week schedule and the other used a four-weekschedule. The three-week schedule used gemcitabine 1250 mg/m², given by30-minute intravenous infusion, on days 1 and 8 of each 21-day cycle.Cisplatin should be administered intravenously at 100 mg/m² on day 1after the infusion of Gemzar®. Dosage reduction with each cycle orwithin a cycle may be applied based upon the amount of toxicityexperienced by the patient.

The four-week schedule used gemcitabine 1000 mg/m², given by 30-minuteintravenous infusion, on days 1, 8, and 15 of each 28-day cycle.Cisplatin at a dose of 100 mg/m² should be administered intravenouslyafter the infusion of Gemzar® on Day 1. Dosage reduction with each cycleor within a cycle may be applied based upon the amount of toxicityexperienced by the patient.

In some embodiments, the chemotherapeutic agent is doxorubicinhydrochloride. Doxorubicin [5,12-Naphthacenedione,10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxylacetyl)-1-methoxy-,hydrochloride (8S-cis)-] is a cytotoxic anthracycline antibioticisolated from cultures of Streptomyces peucetius var caesius (U.S. Pat.No. 3,590,028). Doxorubicin intercalates the base pairs of the DNAdouble helix, thus inhibiting nucleic acid synthesis, inhibitingtopoisomerase II, and producing oxygen radicals. It is a red-orange,crystalline powder, which dissolves easily in water.

When doxorubicin is administered as a single agent, a recommended doseper cycle is 60-75 mg/m² every three weeks. The drug is generally givenas a single dose per cycle; however, it is possible to give the drugdosage per cycle in divided administrations (e.g., day 1 through 3, ordays 1 and 8). Administration of doxorubicin in a weekly regimen hasbeen shown to be as effective as the tri-weekly schedule. Therecommended weekly dosage is 10-20 mg/m². In combination chemotherapy,the recommended dose per three-week cycle is in the 30-60 mg/m² range.

The frequency of administration, dosage amounts, and the duration oftreatment of each of the active agents may be determined depending onseveral factors, e.g., the overall health, size and weight of thepatient, the severity of the disease, the patient's tolerance to thetreatment, and the particular treatment regimen being administered. Forexample, duration of treatment with FTS or the combination of FTS andthe chemotherapeutic agent may last a day, a week, a year, or untilremission of the disease is achieved. Thus, relative timing ofadministration of these active agents is not critical (e.g., FTS may beadministered before, during, and after treatment with thechemotherapeutic agent).

As used herein, the term “effective amount” refers to the dosage(s) ofFTS alone or in combination with the chemotherapeutic agent that iseffective for the treating, and thus includes dosage amounts thatameliorate symptom(s) of the disorder and its associated manifestations,diminish extent of disease, delay or slow disease progression, orachieve partial or complete remission or prolong survival. The averagedaily dose of FTS generally ranges from about 50 mg to about 2000 mg,and in some embodiments, ranges from about 200 mg to about 1200 mg. Theaverage dose of paclitaxel according to its prescribed regimen generallyranges from about 10 mg to about 300 mg, and in some embodiments about10 mg to about 200 mg. The average dose of docetaxel generally rangesfrom about 10 mg to about 130 mg, and in some embodiments about 10 mg toabout 100 mg. The average dose of cisplatin generally ranges from about10 mg to about 170 mg, and in some embodiments about 10 mg to about 120mg. The average dose for carboplatin generally ranges from about 30 mgto about 620 mg, and in some embodiments about 30 mg to about 400 mg.The average dose of gemcitabine generally ranges from about 50 mg toabout 1700 mg, and in some embodiments about 50 mg to about 1000 mg. Theaverage dose of doxorubicin generally ranges from about 10 mg to about130 mg, and in some embodiments about 10 mg to about 100 mg.

In some embodiments, FTS is administered on a daily basis, e.g., each insingle once-a-day or divided doses, while the chemotherapeutic agent isadministered in accordance with its approved dosing schedule. In someembodiments, both drugs may be administered at the same or at differenttimes.

The methods of the present invention may be used for the treatment ofcancer in mammals, particularly humans. The actives may be administeredin accordance with standard methods. In preferred embodiments, FTS isadministered orally. In an oral dosage form, the FTS is typicallypresent in a range of about 50 mg to about 500 mg, and in someembodiments, from about 100 mg to about 300 mg.

In some embodiments, FTS may be administered by dosing orally on a dailybasis for three weeks, followed by a one-week “off period”, andrepeating until remission is achieved. In another embodiment, FTS may beadministered by dosing twice daily and continuing the treatment untilremission is achieved. Parenteral administration may also be suitable.

In preferred embodiments, the chemotherapeutic agent, e.g., paclitaxel,docetaxel, cisplatin, carboplatin, gemcitabine, and doxorubicin, isadministered intravenously. The agent is typically administered as adrip infusion into the vein through a cannula. Agents may also be giventhrough a central line, which is inserted under the skin into a veinnear the collarbone, or into a PICC line which is inserted into a veinin the crook of the arm.

In some embodiments, the administration of FTS with the chemotherapeuticagent may be cyclic and repeated until remission is achieved. Forexample, in one treatment regimen, FTS (200 mg) is administered twicedaily for a period of three weeks followed by a one-week intervalwithout FTS (“off period”) while the chemotherapeutic agent, e.g.gemcitabine (Gemzar®), is administered once weekly (1500 mg) for aperiod of three weeks, followed by a one-week rest period. The treatmentregimen is repeated as many times as needed, e.g., until remission isachieved. Under this regimen, gemcitabine and FTS are administered inthree-week cycles (with increasing or decreasing dose amounts as needed)each separated by a one-week “off period”. Dosage reduction with eachcycle or within a cycle may be applied based upon the amount of toxicityexperienced by the patient. Combination chemotherapy may also beadministered in accordance with standard procedures while dosing withFTS.

In another embodiment, the treatment regimen may entail administrationwith oral FTS (e.g., a capsule or a tablet) continuously withoutinterruption (i.e., without an “off period”) and intravenous cisplatinas a daily infusion for five days every three to four weeks untilremission is achieved. Dosing regimens for administering thechemotherapeutic agent or agents may be administered according tostandard procedures or may be adjusted to meet the particular needs ofthe patient.

Oral compositions for FTS and its analogs for use in the presentinvention can be prepared by bringing the agent(s) into association with(e.g., mixing with) a pharmaceutically acceptable carrier. Suitablecarriers are selected based in part on the mode of administration.Carriers are generally solid or liquid. In some cases, compositions maycontain solid and liquid carriers. Compositions suitable for oraladministration that contain the active are preferably in solid dosageforms such as tablets (e.g., including film-coated, sugar-coated,controlled or sustained release), capsules, e.g., hard gelatin capsules(including controlled or sustained release) and soft gelatin capsules,powders and granules. The compositions, however, may be contained inother carriers that enable administration to a patient in other oralforms, e.g., a liquid or gel. Regardless of the form, the composition isdivided into individual or combined doses containing predeterminedquantities of the active ingredient or ingredients.

Oral dosage forms may be prepared by mixing the active pharmaceuticalingredient or ingredients with one or more appropriate carriers(optionally with one or more other pharmaceutically acceptable additivesor excipients), and then formulating the composition into the desireddosage form e.g., compressing the composition into a tablet or fillingthe composition into a capsule or a pouch. Typical carriers andexcipients include bulking agents or diluents, binders, buffers or pHadjusting agents, disintegrants (including crosslinked and superdisintegrants such as croscarmellose), glidants, and/or lubricants,including lactose, starch, mannitol, microcrystalline cellulose,ethylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, dibasic calcium phosphate, acacia,gelatin, stearic acid, magnesium stearate, corn oil, vegetable oils, andpolyethylene glycols. Coating agents such as sugar, shellac, andsynthetic polymers may be employed, as well as colorants andpreservatives. See, Remington's Pharmaceutical Sciences, The Science andPractice of Pharmacy, 20th Edition, (2000).

Liquid form compositions include, for example, solutions, suspensions,emulsions, syrups, elixirs and pressurized compositions. The activeingredient or ingredients, for example, can be dissolved or suspended ina pharmaceutically acceptable liquid carrier such as water, an organicsolvent (and mixtures thereof), and/or pharmaceutically acceptable oilsor fats. Examples of liquid carriers for oral administration includewater (particularly containing additives as above, e.g., cellulosederivatives, preferably in suspension in sodium carboxymethyl cellulosesolution), alcohols (including monohydric alcohols (including monohydricalcohols and polyhydric alcohols, e.g., glycerin and non-toxic glycols)and their derivatives, and oils (e.g., fractionated coconut oil andarachis oil). The liquid composition can contain other suitablepharmaceutical additives such as solubilizers, emulsifiers, buffers,preservatives, sweeteners, flavoring agents, suspending agents,thickening agents, colorants, viscosity regulators, stabilizers orosmoregulators.

Carriers suitable for preparation of compositions for parenteraladministration include Sterile Water for Injection, Bacteriostatic Waterfor Injection, Sodium Chloride Injection (0.45%, 0.9%), DextroseInjection (2.5%, 5%, 10%), Lactated Ringer's Injection, and the like.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols and mixtures thereof, and in oils. Compositions may also containtonicity agents (e.g., sodium chloride and mannitol), antioxidants(e.g., sodium bisulfite, sodium metabisulfite and ascorbic acid) andpreservatives (e.g., benzyl alcohol, methyl paraben, propyl paraben andcombinations of methyl and propyl parabens).

In order to fully illustrate the present invention and advantagesthereof, the following specific examples/experiments are given, it beingunderstood that the same is intended only as illustrative and in no waylimitative.

EXAMPLE 1 Experimental Design

The purpose of these in vitro and in vivo experiments was to assess theability of FTS, alone and in combination with a chemotherapeutic agent,to impact lung cancer cell integrity and survival. Here, the effects ofthe Ras inhibitor FTS on growth of non-small cell lung carcinoma (NSCLC)cell lines H-1299 [American Type Culture Collection (“ATCC”), CRL-5803),H23 (ATCC, CRL-5800, K-Ras mutation), HTB54 (ATCC, K-Ras mutation), A549(ATCC, K-Ras mutation) and on the growth of lung squamous cell carcinomacell line SK-MES-1 (ATCC, HTB-58) were examined. FTS on tumor cellgrowth in a nude mouse model was also examined. In addition, thecombination of FTS and a chemotherapeutic agent on tumor cell growthinhibition was examined. The primary goal was to determine: (I) whetherFTS induced cell-cycle arrest in A549 cells and also whether FTS inducedgrowth inhibition in all five human lung cancer cell lines; (II) whetherFTS altered cytoskeleton organization in A549 cells; (III) whether FTSinhibited active K-Ras-GTP and inhibited anchorage-independent growth oflung cancer cells in A549 cells; (IV) whether A549 cells were resistantto apoptosis after exposure to a chemotherapeutic agent in the presenceof FTS; (V) whether FTS administered i.p. inhibited tumor growth in bothA549 and HTB-58 (SK-MES-1) nude mouse models and whether oral FTS,alone, and in combination with a chemotherapeutic agent inhibited tumorgrowth in the A549 lung cancer cell nude mouse model; and (VI) whetherincreasing concentrations of FTS sensitized human NSCLC cell lines H1734and H2030 (KRAS mutations) and H1975 and H3255 (EGFR mutations) to celldeath.

The results of the first set of experiments (I) demonstrated that FTSinduced cell cycle arrest in A549 cells. In addition, FTS causeddose-dependent inhibition in A549, HTB54, and H23 cell lines (whichharbor activated K-Ras) and in H-1299 and HTB-58 (SK-MES-1) cell lines(neither of which harbors mutated Ras). Thus, FTS inhibited the growthof tumor cells even when the cells did not harbor mutated Ras genes.Results also indicated that the half-maximal inhibitory concentration(IC₅₀) of FTS ranged between 30 to 75 μm depending on the cell line.

The second set of experiments (II) revealed that A549 cells treated withFTS showed strong actin stress fibers and focal adhesions as comparedwith the control cells. Thus, FTS altered cytoskeleton organization andcell morphology in the A549 cell line.

In the third set of experiments (III), FTS inhibited the development ofA549 human lung cancer cell colonies. Thus, FTS inhibited theanchorage-independent growth of A549 cells. In addition, FTS reduced theamount of K-Ras-GTP in a dose-dependent manner.

The results of the fourth set of experiments (IV) revealed that FTSincreased sensitivity of A549 cells to cytotoxic drugs. Results showedthat the combination of FTS and the chemotherapeutic agent demonstratedthat the combined treatment with both drugs was more effective thantreatment with either drug alone in A549 cells.

In the fifth set of experiments (V), i.p. administration of FTSinhibited tumor growth in A549 and HTB-58 (SK-MES-1) cell nude mousemodels. Thus, FTS (i.p.) inhibited tumor growth as elicited by A549 andSK-MES-1 cells in vivo. In addition, oral administration of FTSinhibited tumor growth in the A549 lung cancer cell nude mouse model.Results also indicated that the combinations of FTS and gemcitabine(oral) were more effective than treatment with either drug alone.

The results of a sixth set of experiments showed that FTS at increasingconcentrations sensitized human NSCLC cell lines H1734 and H2030 (KRASmutations) and H1975 and H3255 (EGFR mutations) to cell death.

Materials and Methods Cell Culture

FTS was provided by Concordia Pharmaceuticals, Inc. (Ft. Lauderdale,Fla.). All cell lines were obtained from American Type CultureCollection (“ATCC”) (Manassas, Va.). A549 cells, non-small-cell lungcarcinoma (CCL, ATCC) cells, were cultured in Kaighn's modification ofHam's F-12 medium containing 1.5 g/l sodium bicarbonate, 10% fetal calfserum (FCS), 100 U/ml penicillin, and 100 μg/ml streptomycin. HTB54 lungcarcinoma cells were cultured in McCoy's 5A medium with 10% FCS, 100U/ml penicillin, and 10 μg/ml streptomycin. HTB-58 (SK-MES-1, ATCC), ahuman lung squamous cell carcinoma cell line, was cultured in Eagle'sminimum essential medium with 2 mM L-glutamine and Earle's BSS, 1.5 g/lsodium bicarbonate, 0.1 mM non-essential amino acids, 1 mM sodiumpyruvate, 10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin. H23(NCI-H23, ATCC), a human non-small-cell lung adenocarcinoma cell line,was cultured in RPMI 1640 medium with 2 mM L-glutamine, 1.5 g/l sodiumbicarbonate, 4.5 g/l glucose, mM HEPES, 1 mM sodium pyruvate, 10% FCS,100 U/ml penicillin, and 100 μg/ml streptomycin. H1299 (NCI-H1299,ATCC), a non-small-cell lung carcinoma cell line, was cultured in RPMI1640 medium with 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 4.5 g/lglucose, 10 mM HEPES, 1 mM sodium pyruvate, 10% FCS, 100 U/mlpenicillin, and 100 μg/ml streptomycin. The cells were plated in 24-wellplates in 1 ml of medium at a density of 5000 cells/well (or 2500cells/well, HTB54) and incubated at 37° C. in a humidified atmosphere of95% air and 5% CO₂. Cells were treated with the indicated concentrationsof FTS (Concordia Pharmaceuticals, Sunrise FL) or with 0.1% Me₂SO₄(DMSO) (vehicle) 24 h after plating and were counted 5 days later. Deadcells were counted after addition of Hoechst 33258 dye (Sigma-Aldrich,St. Louis, Mo.); 1 μg/ml) to vehicle-treated control cultures or tocultures treated for 24 or 48 h with 75 μM FTS. Fluorescence images werecollected 5 min after the dye was added.

In drug combination experiments, cells were grown for 2 days in theabsence or in the presence of 40 μM FTS and were then treated for 4 hwith gemcitabine (100 or 200 nM), cisplatin (50 or 100 nM), doxorubicin(50 or 100 nM), or paclitaxel (2.5 or 5 nM). Live cells were countedafter a further 3 days of incubation with or without FTS. Experimentswere performed twice in quadruplicate.

BrdU Incorporation into DNA

A549 cells were plated on glass cover slips (1.2×10⁵ cells/well in6-well plates) and incubated for 24 h in medium containing 5% FCS. Thecells were then incubated for 24 h with or without 75 μM FTS and thenfor 24 h with 5-bromo-2-deoxyuridine (BrdU) (Zymed BrdU labeling kit,1:100 dilution). Cells were fixed with 4% paraformaldehyde,permeabilized with 0.2% Triton X-100 (BDH, Poole, UK), washed with PBS,blocked with TBS Tween (TBST; 50 mM Tris, pH 7.4, 100 mM NaCl, 0.1%Tween 20) containing 1% bovine serum albumin (BSA), treated sequentiallywith 2 N HCl and 0.1 M sodium borate pH 8.5, and then blocked with goatγ-globulin and washed with TBST-BSA (described above). The cells werethen labeled successively with mouse anti-BrdU antibody (Ab) (Zymed kit;1:50 dilution), biotinylated rabbit anti mouse IgG (5 μg/ml), andCy3-streptavidin (1.5 μg/ml). Cells with BrdU-stained nuclei werecounted under a fluorescence microscope.

FACS Analysis

A549 cells were plated (9×10⁵ cells) in 10-cm plates, incubated for 24 hin medium containing 5% FCS, and then incubated for 24 or 48 h with orwithout 75 μM FTS. The cells were collected, resuspended in PBScontaining propidium iodide (50 μg/ml; Sigma) and 0.05% Triton X-100,and subjected to analysis by a fluorescence-activated cell sorter(FACSCalibur; Becton Dickinson, Los Angeles, Calif.).

Immunofluorescence and Confocal Microscopy

A549 cells were plated on glass cover slips (2×10⁴ cells/well in 6-wellplates), incubated for 24 h in medium containing 5% FCS, and thenincubated for 48 h with or without μM FTS. The cells were fixed andpermeabilized at room temperature by successive incubations with 3.7%formaldehyde (20 min) and 0.2% Triton X-100 in PBS (5 min), then washedfor 5 min with UB buffer (150 mM NaCl, 10 mM Tris pH 7.6, and 0.2%sodium azide in PBS) and blocked with 2% BSA in UB (UBB, 5 min). Thefixed cells were incubated successively with naïve goat IgG for 30 min(200 μg/ml, Jackson ImmunoResearch Laboratories, West Grove, Pa.),anti-vinculin Ab for 1 h (1:400, Sigma-Aldrich), goat anti-mouseCyt-conjugated Ab for 1 h (1:200, Jackson), and rhodamine-labeledphalloidin for 1 h (1:1000, Sigma-Aldrich). Between each of the abovesteps the cells were washed for 30 min with UBB. Lastly, the cover slipswere washed with UB, dried, and mounted onto the slides with Muviol.F-actin (red) and vinculin (green) were visualized with a Zeiss LSM 510confocal microscope fitted with non-leaking green and red fluorescencefilters. Co-localization was assessed using the co-localization functionof the LSM 510 software.

Hoechst Staining Procedures

Hoechst 33258 dye, an ultraviolet light-excitable dye that demonstratesincreased fluorescence when bound to the condensed chromatin ofapoptotic cells, was used to quantify apoptotic cells in cell cultureafter FTS treatments. In tissue culture, cells were seeded at a densityof 20×10⁴ cells in 6-well plates for 24 h. Once cells had reached 70%confluence in normal FCS, the media was changed to low serum media (0.5%FCS for) and FTS was added. Control cells were treated with 0.1% DMSO.Hoechst solution was added to each well for 5-10 min and three picturesfrom each well were taken while using fluorescence microscopy.

Anchorage-Independent Colony Formation Assay in Soft Agar

Noble agar (2% and 0.6%; Difco, Detroit, Mich.) was prepared in waterand autoclaved. The 2% agar was melted in a microwave oven, mixed 1:1with medium (×2 Kaighn's modification of Ham's F-12 medium with 20% FCS,100 U/ml penicillin, and 0.1 mg/ml streptomycin) and poured onto 96-wellplates (50 μl per well) to provide the 1% base agar. The 0.6% agar (5ml) was mixed with 5 ml of medium (×2), containing 8×10⁴ A549 cells, andthe mixture (50 μl) was plated on top of the base agar. The cells wereincubated for 19 days at 37° C. with or without the indicatedconcentrations of FTS (6 wells for the control and for each treatment)and colonies were stained with MTT (1 mg/ml for 4 h). The colonies werethen visualized by light microscopy, imaged, and counted using theImagePro software.

Ras, Rac and Rho Pull-Down Assays and Immunoblotting Procedures

A549 cells were incubated for 24 or 48 h with or without FTS, asdescribed above, and then lysed with lysis buffer as described inHaklai, R., Gana-Weisz, M., Elad, G., et al., Biochemistry 37:1306-14(1998). The apparent amounts of K-Ras-GTP in 0.5 mg protein of totalcell lysates were determined by the glutathione-S-transferase (GST)-RBD(Ras-binding domain of Raf) pull-down assay, as described in(Elad-Sfadia, G., Haklai, R., Ballan, E., Gabius, H. J., Kloog, Y., JBiol Chem 277:37169-75 (2002). The apparent amounts of Rac1-GTP and ofRhoA-GTP, each in 2 mg protein of total cell lysates, were determined,respectively, by pull-down assays with GST-PBD (Rac1-binding domain ofPAK1)-conjugated and GST-Rhotekin BD (Rho-binding domain ofRhotekin)-conjugated beads [Benard, V., Bohl, B. P., Bokoch, G. M, JBiol Chem 274:13198-204 (1999); Fiordalisi, J. J., Keller, P. J., Cox,A. D., Cancer Res 66:3153-61 (2006). The pulled-down GTPases weresubjected to SDS-PAGE followed by immunoblotting with the appropriateantibodies: anti K-Ras (1:30; Calbiochem, La Jolla, Calif.), anti Rac-1(1:2500; Santa Cruz Biotechnology, Santa Cruz, Calif.), or anti RhoA(1:700; Upstate Biotechnology, Lake Placid, N.Y.). Immunoblots wereexposed to 1:2500 peroxidase-goat anti-mouse IgG. Levels of phospho-ERKand phospho-Akt were determined by immunoblotting [Haklai, R.,Gana-Weisz, M., Elad, G., et al., supra.] using rabbit antiphospho-ERK1/2 Ab (Santa Cruz Biotechnology, Santa Cruz, Calif.) andrabbit anti phospho-Akt Ab (Cell Signaling, Beverly, Mass.). Proteinbands were visualized by enhanced chemiluminescence and quantified bydensitometry using ImageJ computer software (National institutes ofHealth, Bethesda, Md.).

Animal Studies

Nude mice (6 weeks old) were housed in barrier facilities on a 12-hlight/dark cycle. Food and water were supplied ad libitum. On day zero,A549 or HTB-58 cells (5×10⁶ cells in 0.1 ml PBS) were implantedsubcutaneously (s.c.) just above the right femoral joint. After 5 or 11days the mice were separated randomly into control groups that hadreceived only the vehicle and FTS-treated groups. Daily FTS treatmentswere administered either intraperitoneally (i.p.) or orally. Tumorvolumes or weights were determined as described in Barkan, B.,Starinsky, S., Friedman, E., Stein, R., Kloog, Y., Clin Cancer Res12:5533-42 (2006). Gemcitabine treatment (36 mg/kg, i.p.) wasadministered every 4 days.

Results I. FTS Inhibited the Growth of A549, H-1299, H23, HTB54, andHTB-58 (SK-MES-1) Human Lung Cancer Cells.

The tumor cell lines of the present study were originally derived fromhuman lung epithelial cells and are representative of lung cancers andits associated manifestations. Here, we examined the impact of Rasinhibitor FTS on growth of non-small cell lung carcinoma cell lines A549(K-Ras mutation), H23 (K-Ras mutation), and H-1299. We also examined theimpact of FTS on the growth of HTB23 lung epidermoid carcinoma cell lineand on lung squamous cell carcinoma cell line HTB-58 (SK-MES-1).

To investigate the effect of FTS on lung cancer cell proliferation, wefirst incubated A549 cells that harbor the activated K-ras gene mutatedat codon 12. A549 cells are commonly used as a model for drug screening.Incubation of the cells with 75 μM FTS for 48 h inhibited theincorporation of BrdU into their DNA by 56.7±17.4% relative tovehicle-treated control cells (P<0.05) (FIG. 1). Typicalphotomicrographs of control and 75 μM FTS-treated A549 cells (72 h)showed that FTS induced a reduction in cell number and altered themorphology of the cells (FIG. 2). Increasing concentration of FTSinhibited A549 cell growth at a dose dependent rate, with a decrease of50% at 40 μM FTS. The number of cells in the FTS-treated cultures wasdetermined by direct counting of A549 cells grown for 6 days in thepresence of FTS and was expressed as a percentage of the number recordedin the controls. Data were means of 12 counts ±SD. *P<0.01, **P<0.0005,compared to control (FIG. 3). In another set of experiments, cells werealso treated for 24 and 48 h with FTS and collected for FACS analysis(FIG. 4). The apoptotic population of cells (indicated in the FACSanalysis as sub-G1) was 3.8% at 24 h and 8.4% at 48 h in cells treatedwith 75 μM FTS, compared to 1.0% and 2.8%, respectively, in controlcells (FIG. 4). The results of these experiments also showed that FTScaused a reduction in the G1 population of cells but not in that of G2/Mcells. Cells treated with FTS for 24 h and 48 h showed reductions in G1of 5.6% and 19%, respectively (FIG. 3). Thus, FTS induced cell-cyclearrest in A549 cells, resulting in inhibition of cell growth.

The growth-inhibitory effects of FTS were not limited to the A549 humanlung cancer cells. Similar growth inhibition curves were obtained forH-1299 cells (FIG. 5) and SK-MES-1 cells (FIG. 6), which expressrelatively large amounts of EGF and insulin-like growth factor (IGF)receptors which activate Ras, and for H23 cells (FIG. 7) and HTB54 cells(FIG. 8), which harbor oncogenic K-Ras. The IC₅₀ values ranged between30-75 μM FTS, depending on the cell line (FIG. 9).

II. FTS Altered Cytoskeleton Organization of A549 Cells.

Next, to determine the effects of FTS on the cytoskeleton of A549 cells,the cells were incubated, treated with 75 μM FTS, and stained withrhodamine-labeled phalloidin, which associates with polymeric F-actin,and with anti-vincullin, which associates with focal adhesions. Typicalfluorescence images of control and of FTS-treated cells are shown inFIG. 10. Cells treated with FTS showed strong actin stress fibers andfocal adhesions as compared with the control cells. The untreated cellsexhibited short, thin actin stress fibers and relatively few focaladhesions, whereas the FTS-treated cells exhibited long, thick stressfibers and a relatively large number of focal adhesions that lookedlarger than those observed in the control cells. Statistical analysisindicated that more than 80% of the cells in the FTS-treated cultureshad undergone changes in cell morphology. These results combined withthe growth-inhibitory effects of FTS observed in lung cancer cell linessuggested that the FTS had, at least, partially reversed the transformedphenotype of the cells. Moreover, these results are consistent with theprevious experiments that demonstrated an observed change in A549 cellmorphology (FIG. 1).

III. FTS Inhibited Anchorage-Independent Growth of A549 Cells.

To determine whether active K-Ras-GTP and its prominent downstreamsignals to ERK and Akt were inhibited in A549 cells, and if so, whetherthe anchorage-dependent growth of the cells was also affected, twoexperiments were performed. First, A549 cells were incubated in theabsence and in the presence of various concentrations of FTS andK-Ras-GTP, phospho-ERK and phospho-Akt levels were measured. FTS reducedthe amount of K-Ras-GTP in a dose-dependent manner with no significanteffect on the total amount of Ras (FIG. 11A). The reduction in K-Ras-GTP(mean±SD) was 23±15.3%, 37±3.7% (P<0.01), and 46±1.9% (P<0.002),respectively, in cells treated with 25 μM, 50 μM, and 75 μM FTS. Theeffective concentration range (50-75 μM) for the reduction in K-Ras-GTP(FIG. 11A) was similar to that required for the inhibition of cellgrowth (FIG. 3). FTS also reduced the levels of phospho-ERK andphospho-Akt causing 33±2% and 58±6% inhibition, respectively (FIG. 2).

The effect of FTS appeared to be specific to the Ras protein, since ithad no effect on the amount of the prenylated active Racl-GTP protein asdetermined by a specific Racl-GTP pull-down assay (FIG. 11B). Moreover,using a specific pull-down assay for prenylated active RhoA-GTP, FTSinduced a significant increase of 2±0.2 fold (P<0.002) in RhoA-GTP (FIG.11C). Thus, while FTS did not reduce the total amounts of the threeGTPases (K-Ras, Rac-1, and RhoA), it clearly had a selective inhibitoryeffect on active K-Ras. The observed increase in RhoA-GTP is consistentwith the observed increase in stress-fiber formation and focal adhesionassembly (FIG. 10).

Next, to determine the effect of FTS on the anchorage-independent growthof A549 cells, a soft agar assay was performed. The cells were seeded insoft agar and treated with increasing concentrations of FTS 0 μM, 50 μMand 100 μM (FIGS. 12A-12B). Control cells were treated with 0.1% Me₂SO₄(DMSO). FTS inhibited A549 cell growth in soft agar by 27±5.5% and58±21% at 50 μM and 100 μM FTS, respectively. Thus, FTS inhibited theanchorage-independent growth of A549 cells.

IV. Combining FTS With a Chemotherapeutic Agent Enhanced Cell Death inHuman Lung Cancer A549 Cells.

To determine whether A549 cells were resistant to apoptosis, anexperiment to examine the survival of human lung cancer A549 cells afterexposure to a chemotherapeutic agent in the presence of FTS wasperformed. Thus, to determine whether treatment with FTS can increasethe sensitivity of A549 cells to cytotoxic drugs, A549 cells wereincubated for 48 h with DMSO (control) or with 40 μM FTS, then for 4 hwith gemcitabine, cisplatin, doxorubicin, or paclitaxel at the indicatedconcentrations. The cells were then washed and incubated for a further72 h with DMSO or with 40 μM FTS. Live cells were collected and counted.The numbers of cells in the drug-treated cultures, expressed aspercentages of the numbers in the vehicle-treated control, are shown inFIGS. 13A-13D. Values are means±SD. *P<0.05, **P<0.01, compared tovehicle-treated control.

As shown in FIG. 13A, the effects of gemcitabine in the presence of FTScaused an enhanced increase in cell death that was measurably moreeffective than treatment with either drug alone in A549 cells. As shown,FTS alone caused a 25±6.3% reduction in cell numbers (mean±SD) at 40 μM,while gemcitabine alone at 100 and 200 nM had no effect (<11%). Thecombinations of FTS and gemcitabine at 100 and 200 nm enhanced cellnumber reductions of 45±5.3% and 60±5.7%, respectively.

As shown in FIG. 13C, the effects of cisplatin in the presence of FTScaused an increase in cell death that was measurably more effective thantreatment with either drug alone in A549 cells. As shown, FTS alonecaused a 33±9.5% reduction in cell numbers (mean±SD) at 40 μM, whilecisplatin alone at 50 and 100 nM caused reductions of 11±11% and30±12.9%, respectively. The combinations of FTS and cisplatin at 50 and100 nm caused cell number reductions of 47±6.9% and 63±12.7%,respectively.

As with cisplatin, the observed effects of the combinations ofdoxorubicin (FIG. 13B) and of paclitaxel (FIG. 13D) in the presence ofFTS caused an increase in cell death that was measurably more effectivethan treatment with either drug alone in A549 cells.

V. FTS Alone and in Combination With a Chemotherapeutic Agent InhibitedTumor Growth in Lung Cancer Cell Nude Mouse Models.

To determine whether FTS inhibited tumor growth in vivo, experimentswere conducted using a nude mouse model. The lung cancer cells wereimplanted s.c. above the right femoral joint and the mice were thentreated with FTS. In a first experiment, the effect of i.p.administration of FTS on tumor growth in A549 cells was assessed.Treatment was started 5 days after cell implantation, by which time thetumors were palpable. Tumor volumes were determined 24 days afterimplantation in two groups of mice (n=8) that had received daily i.p.administration of either the vehicle (control) or 10 mg/kg FTS.Significant inhibition of tumor growth relative to the control (53.8%,P<0.05) was recorded in the FTS-treated group (FIG. 14A).

In a second experiment carried out with mice implanted s.c. with HTB-58cells (n=7 per group), significant inhibition of tumor growth(76.4±48.8%) was observed in the group treated daily with 10 mg/kg FTSi.p. (FIG. 14B). Tumor volume measured 14 days after cell implantationin that group was 0.02±0.045 cm³ compared to 0.09±0.08 cm³ in thevehicle-treated controls (P<0.05).

In an additional set of experiments, the A549-cell-implanted nude mousemodel was used to examine the effect of orally administered FTS on tumorgrowth. First, cells were implanted as described above and daily oraltreatment with FTS (50 mg/kg; n=6) or vehicle (n=5) was started either11 days (FIG. 14C) or 6 days (FIG. 14D) after implantation. As shown inFIG. 14C, after 16 days of treatment the tumor weights (mean±SD) inFTS-treated and control mice were 0.4±0.19 g and 0.9±0.39 g,respectively, representing a significant inhibition of 53.7±19.1% intumor growth (P<0.025) in the FTS-treated mice. Next, the effects oforally administered FTS, alone or in combination with gemcitabine, onA549-cell tumor growth was examined (FIG. 14D). Six days after cellimplantation, mice were divided into four groups (n=8 per group) andtreated orally with vehicle alone (control), FTS alone (60 mg/kg),vehicle and gemcitabine (36 mg/kg, i.p. every 4 days), or FTS andgemcitabine. Treatments with gemcitabine began 1 week after FTStreatment was started. Consistent with the results of the firstexperiment (FIG. 14C), oral FTS treatment caused a significantinhibition in tumor growth; tumor weights in the mice treated withvehicle only (control) and with FTS only (mean±SD) were 0.90±0.40 g and0.49±0.15 g, respectively (46.2±16.3% inhibition, P<0.02; FIG. 14D). Asignificant reduction in tumor weight (P<0.015) was also observed in afifth group of mice treated with gemcitabine alone (FIG. 14D). Thecombined effect of gemcitabine and FTS treatments were more effectivethan the effect of each treatment alone. Thus, the result reinforces theresults of the in vitro experiments indicating that combined treatmentwith the two drugs was more effective than treatment with either of thedrugs alone.

VI. FTS Alone Sensitized Human NSCLC Cell Lines H1734, H2030, H1975, andH3255 to Cell Death.

To determine whether FTS sensitized other human NSCLC cell lines to celldeath, experiments were conducted on cell lines H1734 and H2030 (KRASmutations) and H1975 and H3255 (EGFR mutations). The four cell lineswere grown in increasing concentrations of FTS (dissolved in DMSO).After 96 hours, viable cells were quantified using an Alamar blue assay.Results are the mean±standard error of three independent experiments, inwhich there were 3 replicates of each condition, as shown in FIG. 15.

The publications cited in the specification, patent publications andnon-patent publications, are indicative of the level of skill of thoseskilled in the art to which this invention pertains. All of thesepublications are herein incorporated by reference to the same extent asif each individual publication were specifically and individuallyindicated as being incorporated by reference.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the appended claims.

1. A method of treating a human afflicted with lung cancer, comprisingadministering to the human an effective amount of FTS or an analogthereof as represented by the formula:

wherein R¹ represents farnesyl or geranyl-geranyl; R² represents thegroups COOR⁷, CONR⁷R⁸, wherein R⁷ and R⁸ are each independentlyhydrogen, alkyl or alkenyl, and COOM wherein M is a cation; R³, R⁴, R⁵and R⁶ are each independently hydrogen, alkyl, alkenyl, alkoxy, halo,trifluoromethyl, trifluoromethoxy, or alkylmercapto; and X represents S;or a pharmaceutically acceptable salt thereof, and a chemotherapeuticagent.
 2. The method of claim 1, wherein the human afflicted with lungcancer is administered FTS.
 3. The method of claim 1, wherein the humanafflicted with lung cancer is administered an analog of FTS which isGGTS.
 4. The method of claim 1, wherein FTS or its analog or apharmaceutically acceptable salt thereof is administered orally.
 5. Themethod of claim 1, wherein the chemotherapeutic agent is administeredintravenously.
 6. The method of claim 1, wherein the chemotherapeuticagent is vinorelbine.
 7. The method of claim 1, wherein thechemotherapeutic agent is doxorubicin.
 8. The method of claim 1, whereinthe chemotherapeutic agent is paclitaxel.
 9. The method of claim 1,wherein the chemotherapeutic agent is docetaxel.
 10. The method of claim1, wherein the chemotherapeutic agent is a platinum based drug or ananalog thereof.
 11. The method of claim 10, wherein the platinum baseddrug is cisplatin.
 12. The method of claim 10, wherein the platinumbased drug is carboplatin.
 13. The method of claim 1, wherein thechemotherapeutic agent is pemetrexed.
 14. The method of claim 1, whereinthe chemotherapeutic agent is doxorubicin.
 15. The method of claim 1,wherein the chemotherapeutic agent is etoposide.
 16. The method of claim1, wherein the chemotherapeutic agent is topotecan.
 17. The method ofclaim 1, wherein the chemotherapeutic agent is vinblastine.
 18. Themethod of claim 1, wherein the chemotherapeutic agent is vindesine. 19.The method of claim 1, wherein the chemotherapeutic agent is ifosfamide.20. The method of claim 1, wherein the chemotherapeutic agent ismitomycin.